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Updated: Aug 9, 2026
The Soft Agar Colony Formation Assay
Published on: October 27, 2014
Anchorage-independent growth of Ewing sarcoma cells under serum-free conditions is not associated with stem-cell like
Katharina Leuchte1, Bianca Altvater1, Simeon Hoffschlag1
1Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster, D-48149 Muenster, Germany.
Abstract:
Novel treatment strategies for Ewing sarcoma aim to eliminate residual tumor cells that have maintained the capacity to reinitiate tumor growth after intensive conventional therapy. Preclinical models that more closely mimic in vivo tumor growth than standard monolayer cultures are needed. Sphere formation under anchorage-independent, serum-free conditions has been proposed to enrich for cells with tumor-initiating, stem cell-like properties in various solid cancers. In the present study, we assessed the phenotype and functional stem cell characteristics of Ewing sarcoma spheres. Spheres were generated under serum-free culture conditions from four Ewing sarcoma cell lines and four relapse tumor biopsies. Standard monolayer cultures were established as controls. Median levels of surface expression of the Ewing sarcoma marker CD99 as well as the supposed stem cell marker CD133 and the neural crest marker CD57 were comparable between spheres and monolayers. Ewing sarcoma spheres from individual tumors failed to continuously self-renew by secondary sphere formation. They contained variable proportions of side populations (SPs). Sphere culture did not enhance the in vivo tumorigenicity of Ewing sarcoma cells in a murine xenograft model. We conclude that sphere formation under serum-free conditions is not a reliable tool to enrich for cells with stem cell characteristics in Ewing sarcoma. By mimicking the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases, in vitro sphere growth may still add value as a preclinical tool to evaluate the efficacy of novel therapeutics.
Insights
Sphere formation does not reliably enrich for stem cell-like properties in Ewing sarcoma (a rare bone cancer). However, in vitro sphere growth may still aid in testing new Ewing sarcoma therapeutics.
Area of Science:
- Oncology
- Cancer Biology
- Stem Cell Research
Background:
- Novel Ewing sarcoma treatments target residual tumor cells with self-renewal capacity.
- Preclinical models are needed to better mimic in vivo tumor growth.
- Sphere formation is a proposed method to enrich for tumor-initiating, stem cell-like properties.
Purpose of the Study:
- To assess the phenotype and stem cell characteristics of Ewing sarcoma spheres.
- To determine if sphere culture enriches for Ewing sarcoma stem cell-like properties.
- To evaluate sphere culture as a preclinical model for Ewing sarcoma.
Main Methods:
- Ewing sarcoma spheres were generated under serum-free conditions from cell lines and tumor biopsies.
- Sphere and monolayer cultures were compared for surface marker expression (CD99, CD133, CD57).
- In vivo tumorigenicity was assessed using a murine xenograft model.
Main Results:
- Surface marker expression was comparable between spheres and monolayers.
- Ewing sarcoma spheres did not demonstrate continuous self-renewal.
- Sphere culture did not enhance in vivo tumorigenicity in xenograft models.
Conclusions:
- Sphere formation under serum-free conditions is not a reliable method to enrich for stem cell characteristics in Ewing sarcoma.
- In vitro sphere growth may serve as a valuable preclinical model for evaluating novel Ewing sarcoma therapeutics due to its mimicry of micrometastasis growth.
